Antibodies are composed of two Fab regions that are connected by a flexible hinge-region to the Fc. While the Fab mediates recognition and binding of the antigen, two important functions of the Fc are to mediate effector function by engagement with Fcγ receptors (1) and to confer long serum half-life by binding to the salvage receptor, FcRn (2). In particular, the slow pharmacokinetics of IgG contribute to the success of antibodies as therapeutics as it enables less frequent dosing compared to other biotherapeutics. Consequently, the majority of approved therapeutic antibodies have full-length IgG format. Unlike IgG, the serum half-life of an isolated Fab fragment is short (3) and such property is required for indications when short plasma half-life is desired as with three FDA approved Fab molecules (4). One therapeutic Fab molecule directed against platelet surface receptor GPIIb/IIIa (abciximab, REOPRO®) is commercially produced by proteolytic cleavage with papain (5), which is the original method of Fab production (6). With the advances in molecular cloning, recombinant expression of antibody fragments has become an attractive route to generate Fab molecules as exemplified by the second approved Fab therapeutic, anti-VEGF (ranibizumab, Lucentis®) (7) and the recently approved Fab against dabigatran (idarucizumab, Praxbind®) (33). Fab molecules are advantageous, for example, when transient systemic activity that does not persist past dosing is desired or when administration and activity are localized to a peripheral compartment such as the eye.
Many proteases against the antibody hinge region have been implicated as the mechanism by which pathogens and tumor cells attempt to evade the host immune response (13). Resulting C-terminal neoepitopes, however, are eventually recognized by the immune system and anti-hinge antibodies (AHA) are generated. Such autoantibodies to the upper-hinge region of the Fab and the lower-hinge region of the F(ab′)2 have been shown in several studies (17-21). These pre-existing AHA titers vary from donor to donor (20) and it may represent past and current exposure to such neoepitopes. In certain instances, AHA can act as surrogate Fc and restore effector function of proteolytically inactivated antibodies (22). As one rationale for using a Fab or F(ab′)2 molecule as the therapeutic format is to eliminate effector function, it would be undesirable to have effector function reinstated by pre-existing AHA and risk any potential safety concerns. Accordingly, there is a need in the art for novel Fab and F(ab′)2 molecules that have reduced or no reactivity with pre-existing AHA in human serum to, inter alia, potentially provide a superior safety profile in a therapeutic setting by minimizing immune responses following drug treatment.